| Due to the immunosuppressive caused by the viral infection of reticuloendotheliosis virus(REV),the reticuloendotheliosis(RE)is still causing great harm to the poultry industry.In order to effectively reduce the harm of REV to poultry industry,it is urgent to understand the detailed mechanism of REV leading to immunosuppression in chickens.In this study,one-day-old SPF chicks were used as the study subjects.Experimental animals were randomly divided into REV infection group and control group.The REV-infected group were injected with 500 ?L of REV dilution fluid in the abdominal cavity,the control chicks were injected intraperitoneally with500 ?L of sterile physiological saline and collected samples at 7,14,21,28,and 35 days after infection.The dynamic changes of lymphocyte cycle and apoptosis in immune organs of chicks were respectively detected by PI and Anexin V-FITC/PI staining method.The CCK8 method was used to detect the changes of lymphocyte proliferative ability in immune organs.RT-PCR technique was used to detect the dynamic change of REV copy number and the expression of Cyclin D1,Cyclin E,CDK2 and CDK4 mRNA in immune organs.The dynamic changes of Cyclin D1 and Cyclin E protein levels were detected by Western Blot.It provides an important reference for elucidation immunopathogenesis and control REV.The main results are as follows:(1)Viruses exist in the thymus of one-day-old SPF chicks infected with REV from 7 to 28 days,no viruses exist in the spleen from 7 to 35 days at the experiment.However,the viruses exist in the bursa of Fabricius from 7 to 28 days.No viruses exist in the others time.(2)The number of apoptotic lymphocytes in thymus of one-day-old SPF chicks infected with REV were significantly(P<0.05,P<0.01)higher than control chicks at 14 to 21 days,the number of apoptotic lymphocytes in spleen were significantly greater than control chicks at 21days(P<0.05),the number of apoptotic lymphocytes in bursa of Fabricius was significantly(P<0.05,P<0.01)higher than control chicks at 14 to 28 days,no significant difference(P>0.05)in the others time.(3)The proliferation of T lymphocytes in thymus of one-day-old SPF chicks infected with REV were significantly(P<0.05,P<0.01)lower than the control chicks at the 14 to 28 days.The proliferation of T lymphocytes in spleen of one-day-old SPF chicks infected with REV were significantly(P<0.05)higher than the control chicks at the 7 days and significantly lower(P<0.05)at 21-28 days,and the proliferation of spleen B lymphocytes were significant(P<0.05,P<0.01)lower than control chicks.The proliferation of bursal B lymphocytes were significantly(P<0.05)higher than control chicks at 7 days and significant(P <0.05,P <0.01)lower at 21 to35 days after REV infection,the others time was no statistical difference(P> 0.05).It was shown that the proliferation of lymphocytes in immune organs of one-day-old SPF chicks infected with REV,whose overall trend was increased at early time and then decreased.(4)After one-day-old SPF chickens were infected with REV,the number of thymic lymphocytes in the G0/G1 phase increased significantly(P<0.05)at the 14 days,the lymphocytes of S and G2/M phase were decreased and the results were opposite on the 28 days.The number of splenic lymphocytes at G0/G1 phase were lower than control chicks on the 14 to21 days and the lymphocytes of S and G2/M phases were higher,whereas the results on the 28 days were opposite.The number of bursa lymphocytes in the G0/G1 phase at the 21 days were higher than the control chicks,The lymphocytes of S and G2/M phases were decreased and the results were opposite on the 28 days.(5)After one-day-old SPF chicks were infected with REV,the expression of Cyclin D1 mRNA in thymus was significantly lower(P<0.05)than the control chicks at 14 days and significantly higher(P<0.01)at 35 days.The expression of Cyclin E mRNA in thymus was significantly decreased(P<0.05,P<0.01)on the 7 to 14 days and significantly increased(P<0.05)on 21 and 35 days.The expression of CDK4 and CDK2 mRNA in the thymus were significantly decreased on the 14 days(P< 0.05)and on the 28 th day,the expression of CDK2 mRNA was significantly(P<0.05)increased.The expression of Cyclin D1 mRNA in the spleen was significantly higher(P<0.01)than the control chicks on 14 days,and significantly decreased(P<0.05,P<0.01)at 28 to 35 days.Compared with the control chicks,the expression of Cyclin E mRNA was significantly increased at the 14 to 21 days(P<0.05,P<0.01)and significantly decreased on 28 days(P<0.01).The expression of CDK4 and CDK2 mRNA were respectively significantly increased at 7,14 and 14 days(P<0.05).The expression of Cyclin D1 mRNA in bursa of Fabricius was significantly(P<0.05)higher than control chicks on days 14 and 35 and was significantly(P<0.05)decrease at 21 to 28 days.The expression of Cyclin E mRNA in bursa of Fabricius was significantly increased(P<0.05)at 7 days and significantly decrease(P<0.05,P<0.01)at 21 to 28 days.The expression of CDK4 mRNA in bursa of Fabricius were significantly increased(P<0.05)at 14 days and the expression of CDK2 mRNA was significantly(P<0.05)decreased on the 28 th day,no significant difference(P>0.05)in the others time.(6)After 1-day-old SPF chicks were infected with REV,Cyclin D1 and Cyclin E protein levels in the thymus were significantly(P<0.05,P<0.01)lower than the control chicks at 21 to28 days and 14 to 21 days,respectively,but the content of Cyclin E protein increased significantly(P<0.01)at 35 days.The protein levels of Cyclin D1 and Cyclin E in the spleen were significantly(P<0.05,P<0.01)higher than the control chicks at 14 to 28 days and 14 to 21 days,also significantly lower(P<0.05,P<0.01)than the control chicks at 35 days and 28 to 35 days,respectively.The contents of Cyclin D1 and Cyclin E proteins in the bursa of Fabricius were significantly(P<0.05,P<0.01)increase at 14 days and 14 to 21 days,also were significantly decrease(P<0.05,P<0.01)at 28 to 35 days and 35 days.Through detecting the above indicators,this research has systematically studied the relationship between the inhibition of immune function and the cell cycle and apoptosis of lymphocytes in immune organs of SPF chickens infected with REV,further elucidating the pathogenic mechanism of REV to provides direction for the prevention of RE. |
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